Studies of the biology of Borrelia burgdorferi (Bb) and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward genetic manipulation of Bb we have developed a method of gene inactivation by allelic exchange, using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We would like to investigate the potential roles of genes located on a 26-kb circular plasmid (cp26) of Bb in environmental sensing and adaptation to the tick vector and mammalian host. We have inactivated several cp26 genes in a non-infectious, laboratory-adapted Bb strain, with the intention of subsequently moving these mutations into an infectious isolate. To date, we have been unsuccessful in transforming infectious Bb using the standard protocol and so have been unable to directly test the effects of these mutations in the infectious cycle. We are modifying the procedure and trying other methods of transformation in order to overcome this technical hurdle. We have tested additional genetic markers with the aim of developing more or better tools for selection and screening of transformants. The ability to perform routine genetic manipulations in Bb will greatly facilitate our studies on adaptation and variation of the spirochete, as well as the diverse research objectives of other investigators in the field.